Treatment of bleeding with low half-life fibrinogen

ABSTRACT

The invention provides low-half life fibrinogen with reduced sialyation as a result of recombinant expression or enzymatic and chemical removal. The low-half life fibrinogen is useful in treating or effecting prophylaxis of bleeding particularly in situations of an acute nature in which a high initial dose and rapid decline to normal or below normal levels is desirable.

BACKGROUND OF THE INVENTION

Fibrinogen, the main structural protein in the blood responsible for theformation of clots, exists as a dimer of three polypeptide chains; theAα (66.5 kD), Bβ (52 kD) and γ (46.5 kD) are linked through 29disulphide bonds. The addition of asparagine-linked carbohydrates to theBβ and γ chains results in a molecule with a molecular weight of 340 kD.Fibrinogen has a trinodal structure, a central nodule, termed the Edomain, contains the amino-termini of all 6 chains including thefibrinopeptides (Fp) whereas the two distal nodules termed D domainscontain the carboxy-termini of the Aα, Bβ and γ chains. Fibrinogen isproteolytically cleaved at the amino terminus of the Aα and Bβ chainsreleasing fibrinopeptides A and B (FpA & FpB) and converted to fibrinmonomer by thrombin, a serine protease that is converted from itsinactive form by Factor Xa. The resultant fibrin monomers non-covalentlyassemble into protofibrils by DE contacts on neighboring fibrinmolecules.

REFERENCE TO A “SEQUENCE LISTING”, A TABLE, OR A COMPUTER PROGRAMLISTING

The Sequence Listing written in file 397876SEQLIST.txt is 558 bytes andwas created on Feb. 20, 2013. The information contained in this file ishereby incorporated by reference.

Fibrinogen is naturally subject to phosphorylation, sulfation, andglycosylation. Glycosylation is a complex process of post-translationalmodification and has important functions in secretion, protein folding,immunogenicity and clearance of glycoproteins from the bloodstream.Glycoprotein glycans are mainly attached to proteins via an N- or anO-glycosidic bond. N-linked glycosylation occurs to the side-chain groupof an Asparagine (Asn) residue, whereas O-linked glycosylation occurs tothe side-chain group of Ser or Thr. It is well established that humanfibrinogen contains glycans linked to Asn residues in the Bβ and gammachains in the Asn-Arg-Thr (Asn at position 364) and Asn-Lys-Thr (Asn atposition 52) sequence, respectively (Topfer-Peterson, 1976, HoppeSeylers Z Physiol Chem 357:1509; Blomback, 1973, J. Biol. Chem248:5806).

The current standard of care for patients with fibrinogen deficienciesinvolves replacement therapy with human fibrinogen containingpreparations such as plasma-derived fibrinogen (pdFIB), or fresh-frozenplasma and cryoprecipitates, both of which contain pdFIB.

Although therapy with human fibrinogen containing preparations can beeffective at controlling bleeding, pathologic thromboses with serioussequalae are well-known complications of such infusions (Lak, Br JHaematol, 1999 October; 107(1):204-6). Often the abnormal clots canoccur after the initial bleeding episode has been treated (Pati, Surg.Neurol. 2008 Feb. 22; Matsumoto, Haemophilia 2008 January; 14(1):153-6).In the broader setting of hereditary bleeding disorders, in which thisphenomenon has also been observed, excessive levels of replaced clottingfactors for prolonged durations have been thought to contribute to theseevents (Franchini, Thromb. Haemost. 2004 May; 91(5):1053-5). In the caseof bleeding due to a deficiency of fibrinogen, replacement therapy withpdFIB has been directly implicated as a contributing factor in theetiology of pathologic thromboses (Kreuz, Transfus. Apher. Sci. 2005June; 32(3):247-53).

SUMMARY OF THE INVENTION

The invention relates to a pharmaceutical composition comprising humanfibrinogen having reduced half-life relative to natural plasmafibrinogen, and a pharmaceutically acceptable carrier. Preferably saidfibrinogen has a sialic acid content of 0-2 moles sialic acid per molefibrinogen. The invention also relates to the use of fibrinogen having areduced half-life relative to natural human plasma fibrinogen in themanufacture of a medicament for the treatment or prophylaxis of bleedingin a patient who is bleeding or is at risk of bleeding. Preferably saidfibrinogen is under-sialyated and recombinant or derived from plasma.Furthermore, the invention relates to the use of fibrinogen having areduced half-life relative to natural human plasma fibrinogen in themanufacture of a medicament for the treatment or prophylaxis of bleedingin a patient who is bleeding or is at risk of bleeding, wherein thetreatment is in a regime sufficient to achieve a peak concentration offibrinogen in plasma including administered and endogenous fibrinogenthat is within or above normal levels, and wherein the plasmaconcentration declines to normal or below normal levels within threedays of first administration.

The invention provides methods of treating or effecting prophylaxis in apatient who is bleeding or is at risk of bleeding, comprisingadministering a dose of fibrinogen having a reduced half-life relativeto natural human plasma fibrinogen to the patient. In some methods, thefibrinogen is under-sialyated fibrinogen. In some methods, thefibrinogen is recombinant human fibrinogen. In some methods, thefibrinogen is human fibrinogen from plasma. In some methods, thehalf-life is reduced by a factor of at least 50% relative to that ofhuman plasma fibrinogen. In some methods, the half-life is less than oneday. In some methods, the dose is greater than 1 g. In some methods, thedose is greater than 3 g. In some methods, the dose is greater than 6 g.In some methods, the dose is greater than 10 g. In some methods, thedose is greater than 12 g. In some methods, the dose delivers a peakplasma concentration of recombinant and endogenous fibrinogen greaterthan 2 g/L. In some methods, the peak plasma concentration ofrecombinant and endogenous fibrinogen is greater than 6 g/L. In somemethods, the peak plasma concentration of recombinant and endogenousfibrinogen is greater than 12 g/L. In some methods, the peak plasmaconcentration of recombinant fibrinogen is greater than 1 g/L. In somemethods, the peak plasma concentration of recombinant fibrinogen isgreater than 3 g/L. In some methods, the peak plasma concentration ofrecombinant fibrinogen is greater than 9 g/L. In some methods, the peakplasma concentration of recombinant fibrinogen is greater than 12 g/L.In some methods, a single dose is administered. In some methods,multiple doses are administered. In some methods, the fibrinogen isadministered as a single dose or multiple doses all within a period of24 hours per bleeding episode. In some methods, the bleeding resultsfrom an acute disorder. In some methods, the bleeding results from atraumatic injury. In some methods, the bleeding results from surgery. Insome methods, the bleeding results from an inherited disorder.

The invention further provides a pharmaceutical composition comprisinghuman fibrinogen having reduced half-life relative to natural plasmafibrinogen. Optionally, the human fibrinogen has a sialic acid contentof 0-2 moles sialic acid per mole fibrinogen. The invention furtherprovides methods of treating or effecting prophylaxis in a patient whois bleeding or at risk of bleeding, comprising administering fibrinogenhaving reduced half-life relative to natural human fibrinogen to thepatient in a regime sufficient to achieve a peak concentration offibrinogen in plasma including administered and endogenous fibrinogenthat is within or above normal levels. In some methods, the plasmaconcentration of fibrinogen including administered and endogenousfibrinogen declines to normal or below normal levels within 3 days offirst administration. In some methods, the plasma concentration offibrinogen including administered and endogenous fibrinogen declines tonormal or below normal levels within 2 days of first administration. Insome methods, the plasma concentration of fibrinogen includingadministered and endogenous fibrinogen declines to normal or belownormal levels within 1 day of first administration. In some methods, thepeak plasma concentration of fibrinogen including administered andendogenous fibrinogen is above 2 g/L. In some methods, the peak plasmaconcentration of fibrinogen including administered and endogenousfibrinogen is above 6 g/L. In some methods, the peak plasmaconcentration of fibrinogen including administered and endogenousfibrinogen is above 10 g/L. In some methods, the peak plasmaconcentration of fibrinogen including administered and endogenousfibrinogen is above 12 g/L. In some methods, the peak plasmaconcentration of fibrinogen including administered and endogenousfibrinogen is 2-12 g/L. In some methods, the peak plasma concentrationof fibrinogen including administered and endogenous fibrinogen is within2-4 g/L and the plasma concentration of fibrinogen declines below 2 g/Lwithin 1 day. In some methods, the bleeding results from trauma. In somemethods, the bleeding results from surgery. In some methods, thebleeding results from an inherited disorder. In some methods, thebleeding results from an inherited or acquired deficiency in acoagulation protein. In some methods, the bleeding is a result of organfailure. In some methods, the bleeding is a result of an iatrogenicdisorder. In some methods, the fibrinogen is administered once or atmultiple times occurring within a period of no more than two hours. Insome methods, the fibrinogen is administered multiple times at intervalsno greater than two days.

The invention further provides methods of treating or effectingprophylaxis in a patient who is bleeding or at risk of bleeding,comprising selecting a regime of human fibrinogen based on a reducedhalf-life of the fibrinogen relative to the half life of natural humanfibrinogen from plasma; and administering the regime to the patient. Insome methods, a regime of recombinant human fibrinogen is selected. Insome methods, the regime comprises administering a dose of humanfibrinogen greater than 4 g to the patient. In some methods, the regimecomprises administering the human fibrinogen at intervals of 24 hours orless. In some methods, the regime comprises administering a single doseof the human fibrinogen. In some methods, the regime comprisesadministering multiple doses of the human fibrinogen.

The invention further provides the use of recombinant fibrinogen athigher dose than plasma fibrinogen for an improved efficacy side effectsprofile.

The invention further provides the use of a reduced half-life ofrecombinant fibrinogen relative to plasma fibrinogen in determining aregime to administer the recombinant fibrinogen.

The invention further provides the use of recombinant fibrinogen toconfer a peak concentration of fibrinogen in a patient that is at orabove normal levels, wherein the concentration declines to normal orbelow normal levels within 12 hours.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows three genomic expression vectors were constructedcontaining the α-, β- or γ-gene under control of the αS1-caseinpromoter.

FIG. 2 shows a further exemplary vector was generated by combining thethree α-, β and γ-fibrinogen constructs in one vector (FIB3 construct).Transgenic cows have been generated from both types of vector.Expression levels have ranged from about 1-3 mg/ml in different lines.

FIGS. 3 A, B: Non-reduced and reduced SDS-PAGE showing recombinant- andplasma-derived fibrinogen. Both recombinant (rh-Fbg) and human plasmafibrinogen (h-Fbg) were isolated in the absence or presence of ε-ACA andsubjected to SDS-PAGE (4-20%) analysis. The proteins were visualized bysilverstaining. Non-reduced (left panel) and reduced (right panel)SDS-PAGE: Lane 1 and 3: rh-Fbg and h-Fbg, respectively purified in thepresence of c-ACA. Lane 2 and 4: rh-Fbg and h-Fbg, respectively purifiedin the absence of c-ACA. Arrows on the right of the left panel indicatethe HMW, LMW and LMW′ fraction. Molecular weight markers are indicatedat the left.

FIGS. 4A, B: Western blot analysis of recombinant and human plasmafibrinogen. Samples were run on a 4-20% SDS-PAGE gel under non-reducedand reduced conditions. Proteins were transferred onto nitrocelluloseand the blot was incubated with an anti-fibrinogen antibody. Lanes 1 and3 show rh-Fbg and h-Fbg, respectively purified in the absence of G-ACA;lanes 2 and 4 show rh-Fbg and h-Fbg, respectively purified in thepresence of G-ACA.

FIG. 5: N-linked glycan profile of recombinant and plasma-derivedfibrinogen. Using HPAEC-PAD, the N-linked profile of recombinantfibrinogen (rh-Fbg) and human plasma-derived fibrinogen (h-Fbg) wasestablished and revealed that rh-Fbg contained mainly neutral glycanswhereas h-Fbg contained mainly charged sialylated.

FIG. 6: Polymerization of purified fibrinogen samples. Polymerizationwas initiated by the addition of thrombin (0.025 U/ml) to bothrecombinant (rh-Fbg, isolated in the presence or absence ofε-Aminoaproic acid (c-ACA)) and plasma fibrinogen (h-Fbg; both 0.5mg/ml) preparations at time 0. After 3 min of incubation, polymerformation was followed as change in turbidity at 350 nm in time.

FIG. 7: Factor XIIIa-catalyzed cross-linking of various fibrinogens.Recombinant fibrinogen (rh-Fbg), plasma fibrinogen (h-Fbg) andcommercial obtained plasma-derived fibrinogen (commercial-Fbg), all in afinal concentration of 8 mg/ml were incubated with thrombin (330 U/ml)and Ca²⁺ (25 mM/ml) for 5 min with or without FXIIIa (2 U/ml). Sampleswere dissolved and run under reducing conditions on a 8% SDS-PAGE.

FIGS. 8A, B: Thrombin-catalyzed fibrinopeptides release fromplasma-derived (A, upper panel) and recombinant fibrinogen (B lowerpanel). Both recombinant and plasma-derived fibrinogen (2 mg/ml) wereincubated with thrombin (0.91 NIH/ml) for 30 min at 37 ° C. and thereaction was stopped by the addition of 1.1% TFA. Fibrinopeptide AP, -A,-B were separated by HPLC on a Vydac 218TP52 column using a 0.25%/minacetonitrile gradient starting from 10% to 20.5%.

FIGS. 9A, B: The release of fibrinopeptides A, AP and B fromplasma-derived fibrinogen (H-Fbg-p) and recombinant Fibrinogen (rh-Fbg)after addition of thrombin. Both fibrinogens (2 mg/ml) were incubatedwith thrombin (0.095 NIH/ml) and the reaction was stopped at differenttime points by the addition of 1.1% TFA. Fibrinopeptide AP, -A, -B wereseparated on a Vydac 218TP52 column as described and for each time pointa single analysis was performed. Results are expressed as a % of themaximal release.

FIG. 10: SDS-PAGE showing recombinant- and plasma-derived fibrinogen.Both recombinant (rh-Fbg) and plasma fibrinogen (h-Fbg) were isolated byGPRP-affinity chromatography and subjected to SDS-PAGE (4-20%) analysis.The proteins were visualized by silver staining. Lane 1 and 2:Non-reduced rh-Fbg and h-Fbg respectively. Lane 3 and 4 reduced rh-Fbgand H-Fbg respectively. Molecular weight markers are indicated at theleft.

FIG. 11: Polymerization of GPRP-isolated recombinant andplasma-fibrinogen. Polymerization was initiated by the addition ofthrombin (0.025 U/ml) in the presence of 1 mM EDTA to both fibrinogenpreparations (0.5 mg/ml) and polymer formation was followed as change inturbidity at 350 nm in time. Similar results were obtained whenpolymerization was performed in the presence of 10 mM Ca²⁺.

FIG. 12: Factor XIIIa-catalyzed cross-linking of recombinant fibrinogen(rh-Fbg) and plasma fibrinogen (h-Fbg) isolated according to theGPRP-method. At time 0, thrombin (1 U/ml) was added to a mixture offibrinogen (60 μg/ml) and FXIII (1 U/ml) in the presence of Ca²⁺ (25mM/ml) for 5 min Samples were dissolved and run under reducingconditions on a 8% SDS-PAGE.

FIG. 13: Binding of thrombin to fibrin derived from recombinant (rh-Fbg)and human plasma fibrinogen (h-Fbg). Both rh-Fbg and h-Fbg were isolatedvia GPRP-affinity chromatography and 0.5 mg/ml (final concentration) wasincubated for 30 min at 37° C. with various concentration of thrombin.Clots were spun down by centrifugation and the remaining thrombinactivity was measured by the S-2238 substrate.

FIGS. 14A, B: Clearance of test (A) and control (B) substances.

DETAILED DESCRIPTION

The following terms and definitions are to be understood as follows.

A bleeding episode refers to a period characterized by bleeding in apatient, which is preceded and followed by longer periods withoutbleeding.

A known risk of bleeding means a patient is at a higher risk than thegeneral population of bleeding either by knowledge of subsequentexposure to a particular event or because the patient has an inheritedor acquired condition that predisposes the patient to bleeding episodes.

Natural human fibrinogen means fibrinogen derived from human plasma.Although such fibrinogen can be purified sufficiently for pharmaceuticaluse, it contains trace amounts of other human proteins.

Recombinant fibrinogen means fibrinogen expressed from recombinantconstruct in cell culture, a transgenic animal or in vitro. Unlessotherwise apparent from the context recombinant fibrinogen meansrecombinant human fibrinogen or from fibrinogen from other mammals.Recombinant fibrinogen expressed in nonhuman cells or a nonhuman animalor in a nonhuman in vitro expression system can be made completely freeof other human proteins. Recombinant fibrinogen can also be entirelyfree of pathogens that may be present in human plasma.

Total plasma concentration of fibrinogen refers to the sum of theconcentration of administered fibrinogen and endogenous fibrinogen.

A patient typically means a human but can include other mammals, such ashorses, dogs, cats, sheep, pigs, mice and rats.

Low half-life fibrinogen means fibrinogen having a reduction inhalf-life (i.e., reduced beyond a margin of experimental error inherentin measuring half-life) relative to natural human fibrinogen in the sameassay. The half-life of natural human fibrinogen in humans is about 3days.

Under-sialyated fibrinogen means fibrinogen having a reduced sialiccontent relative to natural human fibrinogen. Natural human fibrinogenhas a sialic content of about 7.7 mole sialic acid per mole fibrinogen.

Endogenous fibrinogen means fibrinogen naturally circulating in theplasma of a patient as distinct from exogenous fibrinogen, which isadministered to a patient.

The invention provides methods of treating or effecting prophylaxis ofbleeding using fibrinogen having reduced half-life relative to naturalplasma fibrinogen (low half-life fibrinogen). The methods are premisedin part on the result that recombinant human fibrinogen can be expressedin a substantially under-sialylated form. This form of fibrinogen has areduced half-life relative to natural human fibrinogen but has similaror improved clotting activity. Some of the disclosed methods are furtherpremised in part on the insight that a reduced half-life is oftenadvantageous in treating or effecting prophylaxis of bleeding. Manydiseases have a chronic nature in which long-term maintenance of steadystate levels of a therapeutic is required. In such disorders,therapeutics with long half-lives are advantageous in reducing dosingfrequency. By contrast, bleeding often has an acute nature. Such isparticularly evident when bleeding occurs as a result of a specificevent, such as trauma or surgery, but is also the case in longer termbleeding disorders in which episodes of bleeding are interspersedbetween periods in which no bleeding occurs. In such situations, lowhalf-life fibrinogen is advantageous because the fibrinogen can beadministered when required to treat or effect prophylaxis of bleedingand rapidly clears from the circulation, decreasing the risk of sideeffects when the fibrinogen is no longer needed. Such administrationoffers an improved efficacy to side effects profile relative to that ofnatural human fibrinogen.

Production of Recombinant Fibrinogen

Fibrinogen is a complex protein consisting of three different chains, α,β and γ, all occurring twice in the total complex. Fibrinogen includesnatural human fibrinogen isolated from human plasma or recombinant humanfibrinogen. Typically, the chains of recombinant fibrinogen are anatural human sequence but truncated or mutated forms having at least 90or 95% sequence identity with natural human forms and retainingfunctional activity (i.e., clotting activity) can also be used. Thethree genes encoding these chains are 6.7, 7.6 and 8.4 kb respectively,and all lie within a 50 kb region on chromosome 4q28. The α-gene liesbehind the γ-gene and is transcribed in the same direction as theγ-gene, whereas the β-gene, which lies downstream of the α-gene, istranscribed in the opposite direction. Each of the fibrinogen chainsincludes a signal sequence that is cleaved in the course ofposttranslational processing and secretion.

Production of biologically active recombinant fibrinogen has been widelydescribed in the scientific and patent literature. For example,recombinant fibrinogen has been produced in human embryonic kidney cells(WO2007/103447), in yeast (WO96/07728), COS cells and other eukaryoticcells (U.S. Pat. No. 6,037,457, EP 1661989). Production of biologicallyactive recombinant fibrinogen has also been achieved in the milk ofseveral transgenic animals, including mice, sheep and cattle (Garner etal., WO95/23868, Velander et al., WO95/22249; Prunkard et al., NatureBiotech 14, 867-71 (1996) and Butler et al. Transgenic Research 13,437-50 (2004), US 2007219352 and WO00/17239).

For transgenic expression, transgenes are preferably designed to targetexpression of a recombinant fibrinogen to the mammary gland of atransgenic non-human mammal harbouring transgene(s) encoding the threefibrinogen chains. The basic approach entails operably linking anexogenous DNA segment encoding a fibrinogen chain including a signalsequence, and a regulatory sequence effective to promote expression ofthe exogenous DNA segment. Typically, the regulatory sequence includes apromoter and enhancer. The DNA segment can be genomic, minigene (genomicwith one or more introns omitted), cDNA, and a YAC fragment. Inclusionof genomic sequences generally leads to higher levels of expression.

Regulatory sequences such as a promoter and enhancer are from a genethat is exclusively or at least preferentially expressed in the mammarygland (i.e., a mammary-gland specific gene). Preferred genes as a sourceof promoter and enhancer include beta-casein, kappa-casein,alphaS1-casein, alphaS2-casein, beta-lactoglobulin, whey acid protein,and alpha-lactalbumin. The promoter and enhancer are usually but notalways obtained from the same mammary-gland specific gene.

Transgenes are introduced into non-human mammals. Most non-humanmammals, including rodents such as mice and rats, rabbits, ovines suchas sheep, caprines such as goats, porcines such as pigs, and bovinessuch as cattle and buffalo, are suitable. Bovines offer an advantage oflarge yields of milk, whereas mice offer advantages of ease oftransgenesis and breeding. Rabbits offer a compromise of theseadvantages. A rabbit can yield 100 ml milk per day with a proteincontent of about 14% (see Buhler et al., Bio/Technology 8, 140 (1990))(incorporated by reference in its entirety for all purposes), and yetcan be manipulated and bred using the same principles and with similarfacility as mice. Nonviviparous mammals such as a spiny anteater orduckbill platypus are typically not employed. In some methods oftransgenesis, transgenes are introduced into the pronuclei of fertilizedoocytes (WO 91/08216). Alternatively, transgenes can be introduced intoembryonic stem cells. Transgenic animals can also be produced by methodsinvolving nuclear transfer (WO 97/07669, WO 98/30683, WO 98/37183, WO98/39416, WO 99/37143).

Analogous strategies can be used to produce fibrinogen in other bodilyfluids of transgenic animals (e.g., urine or blood) or in transgenicplants or plant seeds. Regulatory sequences are selected appropriate forthe cell type in which expression is intended. For example, the promoterfrom the uroplakin gene is suitable for expression in urine. Fibrinogenchains' natural promoters or promoters from coagulation proteins such asprotein C or Factor VIII are suitable for expression in blood.

In general, similar strategies can be used for expression in cellculture except that there is usually more flexibility regarding choiceof promoter and introduction into cells is simpler. Microbes, such asyeast, are one useful system. Saccharomyces is a preferred host, withsuitable vectors having expression control sequences, such as promoters,including 3-phosphoglycerate kinase or other glycolytic enzymes, and anorigin of replication, termination sequences and the like as desired.Insect cells in combination with baculovirus vectors can also be used.

Mammalian tissue cell culture can also be used to express and producebiologically active fibrinogen. A number of suitable host cell linescapable of secreting intact immunoglobulins have been developedincluding CHO, Cos, HeLa, PER.C6® cells and myeloma cell lines.Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter, and anenhancer, and necessary processing information sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Suitable expression controlsequences include promoters derived from immunoglobulin genes, SV40,adenovirus, bovine papilloma virus, or cytomegalovirus.

Irrespective of the expression system or purification method,recombinant fibrinogen is typically purified from its environment (e.g.,milk or cell culture). Several methods for purification of fibrinogenhave been described (EP1115742; EP1115745 describing methods ofpurification by cation exchange and hydrophobic interactionchromatography respectively). Fibrinogen can also be purified by ethanolprecipitation or affinity chromatography as discussed in the Examples.

Recombinant fibrinogen is prepared with reduced glycosylation, and inparticular reduced sialyation, compared with natural human fibrinogenfrom plasma. The level of sialyation of plasma fibrinogen is about(i.e., within a margin or error inherent in measurement) 7.7 mole sialicacid per mole fibrinogen. Thus, under-sialyated fibrinogen ischaracterized by about 0-7 mole sialic acid per mole fibrinogen, andpreferably 0-1, 0-2, 0-3, 0-4, 0-5, 0-6 or 1-2 mole sialic acid per molefibrinogen. Most preferred is a sialic acid content that still rendersthe protein active but with a lower half life than the natural humanfibrinogen. As shown in the Examples, the reduction in sialyation can bethe result of recombinant expression without further manipulation invitro. Under sialyation has also been reported in other proteinsexpressed in cell culture or transgenically. Alternatively oradditionally recombinant fibrinogen can be subject to desialyation byenzymatic or chemical treatment as described further below.

Under-sialyation results in increased clearance via specific receptorsin the liver and consequently decreased half-life, because thedesialylated glycoproteins are recognised by various carbohydratereceptors in the body (Morell et al. (1971) J. Biol. Chem. 246, 1461). Arelative reduction in half-life between under-sialyated fibrinogen andnatural fibrinogen can be shown by pharmacokinetic studies in an animalmodel as disclosed in the Examples. However, absolute half-lives of ahuman protein in a human are usually longer than those in an animalmodel because in an animal model the human protein is subject to animmune response against a non-self protein that would not be present ina human The normal half-life of human plasma fibrinogen whenreintroduced into human plasma is about three days. The half-life ofunder-sialyated recombinant fibrinogen is shorter than that of humanplasma. The extent of shortening depends on the extent ofunder-sialyation but can be less than 75%, 50% or 25% of natural humanfibrinogen. Optionally, under-sialyated human fibrinogen has a half-lifethat is 10-75% of that of natural human fibrinogen and sometimes 20-50%of natural human fibrinogen in humans. Optionally, under-sialyated humanfibrinogen has a half-life in humans of less than 2 days, less than 1day, or less than 12 hours.

Plasma Fibrinogen

Natural human fibrinogen isolated from plasma is commercially availablefrom several sources, including Baxter, Aventis, Alpha Therapeutics,Innovative Research and Sigma Aldridge. Such fibrinogen is extensivelyglycosylated having about 7.7 mol sialic acid per fibrinogen molecules,and has a plasma half-life of about three days. The sialic acid residuescan be removed by either enzymatic or chemical treatment. For enzymatictreatment, the preferred enzyme is sialidase 1 also known asneuraminidase. The enzyme is commercially available from variouscommercial suppliers (e.g., New England Biolabs, Sigma Aldrich) andvarious origins (e.g., human, arthrobacter ureafaciens and Clostridiumperfringens). Neuraminidase is capable of cleaving all non-reducingunbranched N-acetylneuraminic and N-glycolylneuraminic acid residues andalso cleaves branched sialic acids at higher concentrations of enzymeand prolonged incubations. Desialyation can also be performed with acidhydrolysis. For example, trifluoromethanesulphonic acid (Edge, et al.(1981) Anal. Biochem. 118, 131-137) has been used extensively to removecarbohydrate from glycoproteins, while leaving the protein backboneintact. Exemplary conditions for chemical and enzymatic treatment arewell described in the manufacturers' instructions and scientificliterature. However, initial titrations with different durations oftreatment or concentrations of reagents are recommended to achievedesired levels of desialyation (e.g., similar to those shown in thepresent examples). Optionally, the sialyation level can be reduced to anaverage of less than 5, 4, 3, 2, or 1 molecules of sialic acid per molof fibrinogen. Optionally, the sialic content is 0-3 or 0-2 moles sialicacid per mole of fibrinogen. Although not necessary furtherdeglycosylation can be performed after removal of sialic acid residuesusing other deglycosylates, such as PNGase F, B-Galactosidase,Glucosaminidase, and O-Glycosidase, all available from QA Bio.

Bleeding Conditions

The methods and uses of the invention are useful in treating oreffecting prophylaxis of a wide variety of conditions characterized bybleeding, regardless of etiology. Such conditions include bleeding as aresult of trauma (e.g., car crash, battlefield, sports), bleeding as aresult of surgery (e.g., orthopedic surgery, organ transplant surgery,cardiovascular surgery, biopsy, dental procedures). Such conditions alsoinclude bleeding as result of organ failure (e.g., kidney, heart, orliver failure). Increased propensity for bleeding can also be caused bytransfusion of plasma or fluids, which effectively dilutes endogenouscoagulation factors or may exert direct effects on the coagulationsystem. Such diseases also include hereditary or acquired conditionscharacterized by a deficiency in fibrinogen or any other moleculeinvolved in blood coagulation. Such a deficiency may be the result ofrelative lack of such a molecule or presence of normal amounts of such amolecule but of diminished function. Bleeding conditions can also resultfrom vascular defects, thrombocytopenia and thombocytopathia, orexcessive fibrinolytic activity. Some examples of bleeding conditionsinclude hemophilia, Hypoprothrombinemia, von Willebrand's disease,Glanzman's thrombasthenia, Soulier Disease, and Factor XI deficiency.Bleeding is sometimes also sometimes due to unknown causes, as in thecase of idiopathic or iatrogenic bleeding.

In some of the conditions discussed above, such as trauma or surgery,the bleeding typically occurs as an acute condition and, ifappropriately treated, may never recur. In other conditions, such asthose characterized by deficiencies in a coagulation molecule, bleedingepisodes recur periodically through a patient's life but can often byinterspersed by relatively long periods without bleeding. Although insome of the above conditions (e.g., a car crash), bleeding isunforeseeable and can be treated only after it occurs, in otherconditions (e.g., scheduled surgery) bleeding is foreseeable and can betreated prophylactically. Prophylactic treatment can also be useful inpatients having chronic bleeding conditions in periods in which nobleeding is present. In all of the above conditions, the plasma levelsof fibrinogen may or may not be reduced below normal levels. Theinvention methods may, therefore, be applied in settings without anabsolute fibrinogen deficiency.

Treatment Regimes

The invention provides methods of treatment and uses in which themedicament comprising the fibrinogen with reduced half-life ismanufactured and subsequently can be administered to patients who arebleeding (therapeutic treatment) or at known risk of bleeding(prophylaxis). A regime of a dosage, route and intervals ofadministration that inhibits, reduces or terminates bleeding, and/orincreases the speed or strength of clot formation and/or increasessurvival in a patient who is bleeding is referred to as atherapeutically effective regime. A regime of a dosage, route ofadministration and intervals of administration that delays the delays,inhibits and/or prevents bleeding in a patient at known risk of bleedingis referred to as a prophylactically effective regime. In someinstances, therapeutic or prophylactic efficacy can be observed in anindividual patient relative to historical controls or past experience inthe same patient. In other instances, therapeutic or prophylacticefficacy can be demonstrated in a clinical trial in a population oftreated patients relative to a control population of untreated patients.

Low half-life fibrinogen can be used in any of the applications ofnatural fibrinogen and may also be used in other applications wherenatural fibrinogen is not applied. However, preferably the relativehalf-lives of low half-life and natural fibrinogen are taken intoaccount in determining the dose and frequency of administration of lowhalf-life fibrinogen. In some methods, low half-life fibrinogen isadministered in a regime that achieves a higher peak plasmaconcentration of fibrinogen (recombinant plus endogenous plasmafibrinogen) than would normally be employed with natural fibrinogen butwith a faster return to normal or below normal values of fibrinogen(recombinant plus endogenous plasma fibrinogen). The high initial peakconcentration is advantageous in obtaining a rapid and strong responsein treating what is often an acute, and sometimes life-threateningcondition. A rapid decline in concentration is advantageous in resultingin removal of excess fibrinogen after it has played its therapeuticrole, reducing the potential for side effects due to excess fibrinogen,such as thrombosis, stroke, heart attack or ischemia. Alternatively, butless preferably, low half-life fibrinogen can be employed at similardosages and peak concentrations as natural fibrinogen, but administeredat more frequent intervals than natural fibrinogen such that the areaunder the curve (AUC) for low half-life fibrinogen and naturalfibrinogen are similar as is patient response to treatment.

The normal plasma volume in an adult human is about 3 L, and the normalconcentration of plasma fibrinogen is about 2-4 g/L. Thus, each threegrams of low half-life fibrinogen increases the plasma concentration byabout 1 g/L. In patients being treated or prophylaxed, the initial levelof plasma fibrinogen is often below normal levels but can also be withinor even about normal levels. If the level of plasma fibrinogen ismeasured before commencing treatment, the dose of administered lowhalf-life fibrinogen can be adjusted to achieve a desired effect ontotal plasma concentration of fibrinogen. In some methods, the dose isadjusted so the peak concentration of total fibrinogen is within normallevels. In other methods, the dose is adjusted so that the peakconcentration of total fibrinogen is above normal levels. In somemethods, the dose can be adjusted to achieve a peak level of totalplasma fibrinogen of 2-15 or 2-12 g/L. In some methods and usesaccording to the invention, the dose is adjusted to achieve a peak levelof total plasma fibrinogen of greater than 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or 15 g/L. In some methods, the dose is adjusted toachieve a peak level of total plasma fibrinogen of 4-12 g/L, 6-12 g/L,8-12 g/L, 4-15 g/L, 6-15 g/L or 8-15 g/L. In some methods, the dosage issuch as to confer a peak level of low half-life fibrinogen of 1-12 g/Lor 1-15 g/L. In some methods, the dose is such as to confer a peak levelof low half-life fibrinogen of greater than 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or 15 g/L. In some methods, the dose is adjusted toachieve a peak level of low half-life fibrinogen of 4-12 g/L, 6-12 g/L,8-12, 4-15, 6-15 or 8-15 g/L. In some methods, the dose of low half-lifefibrinogen is 1-12 or 1-15 g, or 1-3, 3-6, 6-9 or 9-12 g. In somemethods, the dose of low half-life fibrinogen is at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 g.

Low half-life fibrinogen can be administered in single or multiple dosesto a patient. In some methods, a single dose is administered perbleeding episode, or multiple doses are administered within a relativelyshort period of time, such as a day, 6 hours or 2 hours per bleedingepisode. The administration of a single dose or multiple doses within ashort period is particularly useful in regimes in which the maximum peakconcentration of total fibrinogen is raised to normal or above normallevels. The administration of a single dose or multiple closely spaceddoses in combination with the low half-life leads to a more rapid returnto normal or below normal levels of fibrinogen and reduced potential forside effects from continued presence of administered fibrinogen afterthe desired effects of treatment or prophylaxis have been achieved.Thus, some such regimes are characterized by a peak concentration oftotal plasma fibrinogen higher than would be sought using plasmafibrinogen as a therapeutic agent, but with a more rapid decline tonormal or below normal levels of total plasma fibrinogen than wouldresult from a conventional regime of plasma fibrinogen.

In other methods, particularly useful in therapeutic treatment orprophylaxis of chronic conditions, low half-life fibrinogen isadministered on multiple occasions for extended periods (e.g., a week ora month). In such regimes, the low half-life fibrinogen can beadministered more frequently than would be the case with plasmafibrinogen to maintain a similar mean plasma total fibrinogen contentand similar therapeutic or prophylactic activity to plasma fibrinogen.The interval of dosing for maintaining approximately steady state levelsof low half-life fibrinogen can be from about 1-3 times the half-life oflow half-life fibrinogen. In some methods, levels of total plasmafibrinogen are monitored, and additional dose(s) of low half-lifefibrinogen are administered responsive to a decline in plasmaconcentration below a desired level (e.g., below 2, 3, 4, 5, 6, 7, 8, 9,10, 11 or 12 g/L).

Low half-life fibrinogen can be administered with or without othertreatments for bleeding. Such other treatments include other proteinsinvolved in coagulation, such as factor VIIa, and factor VIII, vonWillebrand's factor, Desmopressin, first aid including applying bandagesor tourniquets, transfusion and surgery to close wounds. Low half-lifefibrinogen is particularly useful when administered within one hourafter bleeding starts. During this period, other treatments,particularly surgery to close wounds, may not be available. Ifappropriate, surgery to close wounds can be performed as soon aspractical after administering low half-life fibrinogen.

In some methods, plasma concentration of fibrinogen is determined beforeand/or after administration of low half-life fibrinogen. Total plasmafibrinogen content can be measured by an ELISA among other methods. Lowhalf-life and natural fibrinogen concentrations can be measuredseparately by ELISA using antibodies specific for the respective formsor one antibody specific for one form in combination with an antibodybinding to both forms. Low half-life and natural fibrinogen can also bedistinguished by HPLC as discussed in the Examples.

The invention relates to a pharmaceutical composition comprising humanfibrinogen having reduced half-life relative to natural plasmafibrinogen, and a pharmaceutically acceptable carrier. In a preferredembodiment, said human fibrinogen has a sialic acid content of 0-2 molessialic acid per mole fibrinogen.

The invention also relates to the use of fibrinogen having a reducedhalf-life relative to natural human plasma fibrinogen in the manufactureof a medicament for the treatment or prophylaxis of bleeding in apatient who is bleeding or is at risk of bleeding. Preferably, saidfibrinogen is under-sialyated. Said fibrinogen is recombinant humanfibrinogen or isolated/derived from plasma.

In a preferred embodiment, the invention relates to said use, whereinthe half-life is reduced by a factor of at least 50% relative to that ofhuman plasma fibrinogen, and preferably wherein the half-life is lessthan one day. Preferably, the dose is greater than 1 g as outlinedabove. In another preferred embodiment of the invention, the dosedelivers a peak plasma concentration of recombinant and endogenousfibrinogen greater than 2 g/L, as outlined above. Preferably, the peakplasma concentration of recombinant fibrinogen is greater than 1 g/L. Inanother preferred aspect, the bleeding results from an acute disorder, atraumatic injury, surgery, or an inherited or acquired disorder, such asan inherited or acquired deficiency in a coagulation protein.

The invention also relates to the use of fibrinogen having a reducedhalf-life relative to natural human plasma fibrinogen in the manufactureof a medicament for the treatment or prophylaxis of bleeding in apatient who is bleeding or is at risk of bleeding, wherein the treatmentis in a regime sufficient to achieve a peak concentration of fibrinogenin plasma including administered and endogenous fibrinogen that iswithin or above normal levels, and wherein the plasma concentrationdeclines to normal or below normal levels within three days of firstadministration. Preferably, the peak plasma concentration of fibrinogenincluding administered and endogenous fibrinogen is above 2 g/L. Inanother preferred embodiment, the peak plasma concentration offibrinogen including administered and endogenous fibrinogen is within2-4 g/L and the plasma concentration of fibrinogen declines below 2 g/Lwithin one day. In another aspect of the invention, the regime is suchthat fibrinogen is administered once or at multiple times occurringwithin a period of no more than two hours. Preferably, the regime issuch that fibrinogen is administered multiple times at intervals nogreater than two days.

Pharmaceutical Compositions and Routes of Administration

Low half-life fibrinogen or pharmaceutical compositions containing thesame are usually administered parenterally with intravenous,intraarterial, or subcutaneous administration being preferred.

Low half-life fibrinogen is often administered with one or more otherpharmaceutically acceptable components as a pharmaceutical composition.See Remington's Pharmaceutical Science (15th ed., Mack PublishingCompany, Easton, Pa., 1980). The preferred form depends on the intendedmode of administration and condition of the patient. The compositionscan also include, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent orpharmaceutically acceptable carrier is selected so as not to affect thebiological activity of the combination. Examples of suchdiluents/carriers are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition can include proteaseinhibitors. The pharmaceutical compositions can also include divalentmetal cation ions, which have been reported to improve stability (seeU.S. Pat. No. 7,045,601). Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Chelating agents, such ascitrate or EDTA can also be included. Other components of pharmaceuticalcompositions can include animal, vegetable, or synthetic origin, forexample, peanut oil, soybean oil, and mineral oil.

Compositions can be prepared as lyophilized powders for reconstitutionprior to use or in liquid solution or suspension. Compositions can bestored frozen, on ice or at room temperature.

The low half-life fibrinogen incorporated in such compositions istypically substantially pure, e.g., at least 85, 95 or 99% w/w pure fromundesired contaminants.

EXAMPLES Example 1 Production of Recombinant Fibrinogen

FIG. 1 shows three genomic expression vectors were constructedcontaining the α-, β- or γ-gene under control of the αS1-caseinpromoter. FIG. 2 shows a further exemplary vector was generated bycombining the three α-, β and γ-fibrinogen constructs in one vector(FIB3 construct). Transgenic cows have been generated from both types ofvector. Expression levels have ranged from about 1-3 mg fibrinogen/mlmilk in different lines.

Example 2 Purification of Recombinant Fibrinogen from Bovine Milk

To compare recombinant fibrinogen (rh-Fbg) to natural human fibrinogen,both recombinant human fibrinogen, expressed in bovine milk, and humanfibrinogen from human plasma has been purified via a similarpurification process. For the purification of recombinant fibrinogen(rh-Fbg), milk production was hormonally induced in a transgenic cow(cow 204; Fay), collected twice a day and stored at below −18° C. untilfurther processing. At the start of the purification, milk was thawedand caseins and fat were removed by high speed centrifugation. Next,ε-Amino-caproic acid (ε-ACA) was added to prevent degradation of thefibrinogen by various proteases present in milk and plasma andsubsequently, the whey (milk without caseins and fat) was subjected toethanol precipitation. The precipitate was collected by centrifugation,washed, dissolved and subjected to another ethanol precipitation step inthe absence of c-ACA. Finally, the pellet was dissolved in 20 mM sodiumcitrate pH 7.0, 0.15 M NaCl buffer, filtered over a 0.45 lam filter andstored at below −50° C. The same procedure was followed for humanplasma-derived fibrinogen (h-Fbg). In addition, for both fibrinogenresources, the procedure has been performed in the absence of c-ACA.

Example 3 Structural Characterization of Fibrinogen

Human plasma fibrinogen appears heterogeneous by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and other methods for separation of proteinsby molecular size. Full size, undegraded fibrinogen has a molecularweight of 340 kD (HMW-fibrinogen) and accounts for approximately 50%-70%of total fibrinogen. A second degraded form with a molecular weight of305 kD (LMW) accounts for about 20-40% of total fibrinogen and theresidual amount is LMW′-fibrinogen with a molecular weight of 280 kD.The two degraded forms differ in their Act-chains i.e. the LMW missesone of the two Aα-chains, whereas both Aα-chains are lacking in the LMW′form, Holm, 1985, Thromb Res 37:165. Both rh-Fbg and h-Fbg were analyzedby SDS-PAGE and revealed that rh-Fbg is very similar to purified h-Fbg(FIG. 3). Under non-reducing conditions and in the presence of ε-ACA,both rh-Fbg and h-Fbg display the three characteristic molecular weightbands (HMW, LMW and LMW′) of fibrinogen in a similar ratio. However,rh-Fbg contains a 40 kD band, an impurity that is not seen in h-Fbg. Inthe absence of ε-ACA, the HMW band from rh-Fbg is degraded and reducedSDS-PAGE analysis revealed that the intensity of the α-chain is reduced.This indicates that the degradation resides in the α-keten of rh-Fbg(FIG. 3, right panel, lane 2) and is most likely caused by plasmin. Nosuch degradation was observed in fibrinogen obtained from plasma.

Under reducing conditions, both fibrinogen preparations appear as threepredominant bands that correspond to the Aα, Bβ and γ-chains offibrinogen. However, the recombinant fibrinogen contains an additionaltriplet below the γ-chains which is not present in plasma-derivedfibrinogen. This triplet may be related to the 40 kD impurities observedon non-reduced SDS-PAGE. In addition, an extra protein band becamevisible above the γ-chain of rh-Fbg and h-Fbg, representing most likelythe γ′ variant. This variant represents in human plasma the alternativesplice variant of the γ-chain that contains two sulfated tyrosins.Chung, 1984, Biochemistry 23:4232

To demonstrate that the different bands observed on SDS-PAGE are indeedderived from fibrinogen bands, their identity was confirmed by westernblot analysis. By using a polyclonal antibody that mainly recognizes theα- and β-chains and to a lesser extent the γ-chain, the similaritybetween rh-Fbg and h-Fbg was confirmed (FIG. 4). As shown by SDS-PAGE,fibrinogen isolated in the absence of ε-ACA misses the α-chain, which isconfirmed by western blot analysis.

Based on the appearance on SDS-PAGE and western blot analysis the purityof the recombinant fibrinogen is estimated to be about 85% with mainimpurities around 40 kD.

This 40 kD impurity was recognized by the anti-fibrinogen antibodyindicating that this is a fibrinogen-derived impurity. One possibilityis that this impurity is due to plasmin-mediated degradation offibrinogen. The purity of the plasma, derived fibrinogen was estimatedto be 95% with most likely fibronectin as main impurity.

Using a high-performance anion-exchange chromatography-pulseamperometric detection (HPAEC-PAD) system, Barroso, 2002, Rapid CommunMass Spectrom 16:1320, the glycosylation of rh-Fbg and h-Fbg wascompared. The N-linked glycan profile showed apparent differences inglycosylation between recombinant fibrinogen that contained mainlyneutral glycans, whereas plasma-derived fibrinogen contained mainlysialylated glycans (FIG. 5). This difference in glycosylation pattern iscommonly seen in eukaryotic recombinant expression systems in whichglycoproteins are produced not as a single structural entity but as aset of differently glycosylated variants of the particular polypeptide.The difference in sialylation was confirmed by measuring the sialic acidcontent of the fibrinogen preparations. For rh-Fbg, this was found to be1.5 mol/mol, whereas h-Fbg was almost fully sialyated at 7.7 mol/mol.Whether the difference in glycosylation influences the function of thefibrinogen molecule is not known. However, it has been suggested that adecrease in sialic acid increases the rate of fibrin polymerization,Henschen, 1993, Thromb Haemost 70:42. (see section 4.1).

Example 4 Functional Characteristics of Recombinant Fibrinogen

A: Polymerization and Clottability of Fibrin Monomers

Fibrinogen is converted into an insoluble fibrin clot in three stages.First, thrombin cleaves the amino-termini of the Aα- and Bβ-chains offibrinogen, releasing fibrinopeptides A and B, respectively, andconverting fibrinogen to fibrin monomers. Next, these fibrils align in ahalf-staggered overlap and polymerize to form fibrin strands. The finalstep is a side-to-side association of the polymers resulting in theformation of the gel. By using the polymerization assay, the threestages of fibrin formation can be followed by assessing the followingthree parameters. 1) The lag-phase: this is the time needed to formprotofibrils upon the addition of thrombin. 2) gelation rate: This isthe slope of the curve which illustrates the speed of fibrin formationand 3) the plateau: this is the maximum OD reached and is related to theaverage size of fibrin fibers i.e. the higher the plateau, the thickerthe fibers.

The polymerization of the rh-Fbg and h-Fbg was initiated by thrombin andfollowed in time at 350 nm. As shown in FIG. 6, both rh-Fbg and h-Fbghave a short lag time (˜1 min), indicating that both preparations have asimilar rate in protofibril formation. However, rh-Fbg has a steeperslope and a higher plateau illustrating that the rate of protofibrilsassembly is faster and the fibrils are thicker in rh-Fbg as compared toh-Fbg. In contrast, the rh-Fbg preparation that was isolated in theabsence of ε-ACA and showed degradation, hardly shows anypolymerization. The difference in fiber thickness observed betweenrh-Fbg and h-Fbg may be explained by the difference in sialic contentbetween rh-Fbg and h-Fbg (see section 3.1.3) and may have implicationsfor the fibrinogenolysis process. Thicker fibrils bind more tissueplasminogen activator (tPA), which may result in a faster fibrinolyticrate and the clot will dissolve more rapidly, Gabriel, 1992, J Biol Chem267:24259.

Nevertheless, these data demonstrate that rh-Fbg is fully functional andhighly comparable with h-Fbg-derived fibrinogen.

To further determine the potency of the fibrinogen preparations, thefibrinogen preparations were incubated with an excess amount of thrombinand the % clottability of the different fibrinogens was compared to acommercial obtained fibrinogen (Enzyme Research Laboratories (ERL)).Whereas the clottability of h-Fbg was comparable with commercialobtained fibrinogen (˜95%), the amount of clottable protein present inthe recombinant fibrinogen was with 79% considerably lower (Table 1).This is most likely explained by the presence of the 40 kD impuritypresent in the recombinant fibrinogen preparation (See section 3.1).When the % clottability was corrected for the impurity, the differencesbetween rh-Fbg and h-Fbg are less pronounced (92% vs 94% clottability,respectively).

TABLE 1 Thrombin-mediated clottability of various fibrinogenpreparations. % clottability Average ± SD (n = 4) Recombinant fibrinogen78.6 (92) ± 0.5     Plasma-derived fibrinogen 94.3 ± 0.1 Commercialfibrinogen 93.1 ± 0.2 (ERL) All three fibrinogen preparations wereincubated with an excess of thrombin (2 U/ml) in the presence of Ca²⁺for 20 min at 37° C. The clotted material was spun down and the residualprotein concentration was determined by measuring the absorbance atOD280 nm. The fraction of clotted protein over total protein wascalculated and expressed as % clottability. The numbers between thequotation marks show the % clottability corrected for the impuritiespresent in the preparation. Commercial fibrinogen was obtained fromenzyme research labs (ERL).B. Platelet Aggregation

As a precursor of fibrin formation, fibrinogen plays a major role inhemostatic plug formation but additionally, it functions as an adhesivefor platelets. Platelet aggregation is initiated upon binding offibrinogen to specific sites of the membrane-bound glycoprotein IIb/IIIa(α_(IIb)β3) which subsequently results in the formation of interplateletbridges, Marguerie, 1979, J Biol Chem 254:5357. This binding is aprecondition for aggregation in vivo.

The ability of recombinant fibrinogen to cause platelet activation wasdetermined and compared to that of plasma-derived fibrinogen. Therefore,platelets were isolated from human platelet rich plasma (PRP) bygel-filtration and pre-incubated with different concentrations of thevarious fibrinogens. Platelet aggregation was triggered by the additionof adenosine diphosphate (ADP). ADP binds to its receptor on theplatelet surface, which initiates aggregation, exposure of fibrinogenreceptors, and calcium mobilization. The results show that rh-Fbgsupports blood platelet aggregation to a almost similar extent asobserved for plasma-derived fibrinogen. The mean values of rh-Fbginduced platelet aggregation showed only 10-15% smaller aggregation ascompared to plasma fibrinogen. This is most likely explained by theimpurity present in the recombinant fibrinogen preparation, sincecomparable results are obtained when the results are corrected for thisimpurity. Plasma-derived fibrinogen supported platelet aggregation to asimilar extent as human fibrinogen (ERL), which was used as a control(Table 2).

TABLE 2 ADP-induced platelet aggregation in the presence of differentconcentrations of fibrinogen. Extent of ADP-induced blood plateletaggregation (%) Fbg Fbg Fbg (200 μg/ml) (100 μg/ml) (50 μg/ml)Recombinant 38 ± 9 27 ± 7 18 ± 4 Fibrinogen Plasma-derived  43 ± 10 32 ±6 20 ± 4 Fibrinogen Human 47 ± 7 nd Nd Fibrinogen Platelets wereisolated from human platelet rich plasma (PRP) by gelfiltration(Sepharose 4B) in the presence of apyrase. Next, platelets (2-3 × 10⁸cells/ml; 0.5 mL) were preincubated with different concentrations of thevarious fibrinogens for 2 min at 37° C. in an aggregometer (Chrono-Log,Havertown, PA) under constant stirring. Platelet aggregation wastriggered by the addition of adenosine diphosphate (ADP; 5 μM). nd = notdone.C: FXIII-Mediated Cross-Linking

In addition to fibrinogen, also Factor XIII (FXIII) plays an importantrole in the regulation of the blood coagulation system. Factor XIII isactivated by thrombin, which results in the formation of thetransglutaminase FXIIIa. This enzyme stabilizes, in the presence ofcalcium, the fibrin clot via covalent cross-links between fibrinmolecules. Furthermore, it protects the fibrin from degradation byplasmin. Muszbek, 1996, Crit Rev Clin Lab Sci 33:357. The basicmechanism of FXIIIa-mediated fibrin cross-linking involves both the α-and γ-chains, but not the β-chains, of fibrin. Activated FXIIIaintroduces a number of cross-links between the γ-chains of twoneighboring fibrin monomers followed by the cross-linking of theα-chains. This latter process occurs more slowly and results in theformation of highly cross-linked α-chain polymers. In addition tofibrin, also fibrinogen can be cross-linked by FXIII. This reactionfollows the same pattern as described for fibrin cross-linking, althoughat a slower rate, Sidelmann, 2000, Semin Thromb Hemost 26:605, and alsoresults in the formation of a clot. The FXIIIa catalyzed cross-linkingof fibrin was examined for both the fibrinogen preparations (recombinantand plasma) and compared with commercial obtained plasma-derivedfibrinogen. All fibrinogen preparations were incubated with thrombin inthe presence or absence of FXIII for 5 min, and the reaction productswere analyzed by reduced SDS-PAGE. Furthermore, bands were analyzed bydensitometric analysis and % dimerization was calculated according tothe following formula: % Dimerization=γγ-dimer/(γγ-dimer+γ-monomer). Asshown in FIG. 7, all three fibrinogen preparations showedγγ-dimerization upon incubation with FXIIIA, that was accompanied by areduction of the α- and γ-monomer bands. However, γγ-dimerization (andthe accompanied reduction of the γ-monomer band) was less pronounced inthe rh-Fbg preparation as compared to both plasma-derived preparations(Table 4). The reason for this may reside in the presences of FXIII, asimpurity, in plasma derived preparations as both preparations showedalready γγ-dimer formation without addition of FXIII. Apparently, theamount of residual FXIII, which was determined by photometric analysis(Table 3) in both plasma-derived fibrinogens, is already sufficient forcomplete γγ-dimer formation.

TABLE 3 γγ-dimer formation of fibrinogen derived from various origins.Dimerization (%) +thrombin Residual neat +thrombin & FXIII FXIII (U/mg)Recombinant Fbg 0.0 0.0 62.2 0.00 Plasma-derived Fbg 0.0 99.6 75.6 0.25Commercial H-Fbg 0.0 99.6 99.4 0.19 Bands shown in FIG. 9 were analyzedby densitometric analysis and % dimerization was calculated according tothe following formula: % Dimerization = γγ-dimer/(γγ-dimer + γ-monomer).D: Fibrinopeptide Release

Fibrinopeptides (FP) are released upon binding by thrombin in an orderedmanner with FP-A release prior to FP-B release. Blomback, 1978, Nature275: 501. In addition, the serine in FP-A can be phosphorylated whichresults in the release of FP-AP. When fibrinogen is incubated with ahigh concentration of thrombin, all fibrinopeptides are released. Thistotal fibrinopeptide release can be analyzed by HPLC analysis. Thefibrinopeptide release of both rh-Fbg and h-Fbg were examined andrevealed that both fibrinogens display the expected fibrinopeptideprofile (FIG. 8). The FP-(A+AP) ratio/FP-B did not differ between thetwo types of fibrinogen, indicating that the FP-A and FP-B release ofthe two type of fibrinogens are comparable (Table 4). However, thedegree of phosphorylated FP-A was higher in the rh-Fbg preparation(approximately 75% FP-AP) as compared to h-Fbg (˜30%).

TABLE 4 Area of the fibrinopeptides released from h-Fbg and rh-Fbg. AreaArea Area Area Area FP-AP FP-A FP-B (A/AP) (A + AP)/B h-Fbg 7398062408847 2535823 0.31 1.24 rh-Fbg 548998 27126 589179 18.29 1.10 Rh-Fbghas more FP-AP than FP-A as compared to h-Fbg. The total area of FP-A +FP-AP is about a much as the FP-B area for both types of Fbg's. The % CVof the area is ± 15%.

Next, the rate of thrombin-catalyzed fibrinopeptide release was examinedby measuring the peak area's of FP-A, FP-AP and FP-B and plotted againsttime. As shown in FIG. 9, the release time of FP-A, FP-AP and FP-B forboth fibrinogens are highly comparable. For FP-A/AP release the t₅₀,i.e. time to reach 50% of the maximal release, for rh-Fbg and h-Fbg is3.8 and 3.9 minutes respectively. In accordance to literature, Weisel,1993, J Mol Biol 232:285, FP-B release was found to be slower comparedto FP-A release and t₅₀ was estimated as 10 and 9.3 minutesrespectively. These data indicate the rate of fibrinopeptide release isindependent of the source of fibrinogen and the degree ofphosphorylation.

Example 5 GPRP-Purified Recombinant Fibrinogen

The data reported above were obtained using a recombinant fibrinogenpreparation that has been purified by sequential ethanol precipitation.Although this purified fibrinogen was fully functional, the preparationcontained a 40 kD impurity. As an alternative purification, recombinantfibrinogen was isolated by affinity purification using the tetrapeptideGly-Pro-Arg-Pro (GPRP) (SEQ ID NO:1) immobilized to Fractogel. Kuyas,1990, Thromb Haemost 63:439. The Gly-Pro-Arg sequence is involved in theinitiation of fibrin polymerization by binding to the complementarybinding site of another fibrinogen molecule. Laudano, 1983, Ann N Y AcadSci 408:315. For the isolation of rh-Fbg from cow milk, a defatted milkfraction was loaded onto a GPRP-coupled (SEQ ID NO:1) Toyopearl affinitycolumn Fibrinogen was eluted from the column by lowering the pH. Asimilar procedure was used for plasma-derived fibrinogen.

By using the GPRP (SEQ ID NO:1) method, both recombinant fibrinogen andhuman fibrinogen isolated from milk and plasma respectively. The resultsrevealed that the recombinant fibrinogen preparation had a purity of>95%and did not contain the 40 kD impurity (FIG. 10). Similar results wereobtained for plasma-derived fibrinogen.

Fibrin polymerization of both GPRP-isolated (SEQ ID NO:1) fibrinogens(rh-Fbg and h-Fbg) was initiated by thrombin and followed in time. Asshown in FIG. 11, rh-Fbg has a shorter lag-phase and reaches a higherplateau as compared to h-Fbg. Both preparations show a similar gelationrate. Furthermore, the % clottability of rh-Fbg was 92%, whereas the %clottability of h-Fbg was found to be 98%. These results indicate thatrh-Fbg isolated via the GPRP-isolation (SEQ ID NO:1) method is fullyfunctional and highly comparable to h-Fbg.

Cross-linking of the α- and γ-chains was performed in the presence ofFXIII and thrombin, and the reaction was analyzed by SDS-PAGE. Bothrh-Fbg and h-Fbg showed a similar cross-linking profile (FIG. 12).

Fibrinogen is converted into fibrin by thrombin. This occurs via afibrinogen recognition site in thrombin, known as exosite 1, Fenton,1988, Biochemistry 27:7106, and results in the cleavage and release offibrinopeptides A and B. In addition, also fibrin is able to bindthrombin. This interaction is mediated via two non-substrate bindingsites, one present in the E-domain, and the other in the D-domain, andresults in the inhibition of thrombin. This activity, named antithrombinI, is one of the major mechanisms involved in controlling thrombinactivity. A failure of this mechanism may have pathological consequencesas indicated by the observation that fibrin from certain congenitaldysfibrinogens exhibit reduced thrombin capacity and is associated withsevere thromboembolism. Mosesson, 2003, Thromb Haemost 89:9.

To determine thrombin binding to fibrin derived from GPRP-isolated (SEQID NO:1) rh-Fbg or h-Fbg, both fibrinogens were incubated with thrombinfor 30 min at 37 ° C., the clots were compacted by centrifugation andthe residual amount of thrombin activity in the supernatants isdetermined using the chromogenic S-2238 substrate. As shown in FIG. 13,both recombinant fibrin and h-Fbg are able to bind thrombin. However,the mean % of thrombin binding of rh-Fbg was with 48% less than thethrombin binding capacity of h-Fbg (62%). An explanation for this is notclear.

rh-Fbg, isolated from milk of transgenic cows is structurally andfunctionally highly comparable to plasma-derived human fibrinogen. Basedon SDS-PAGE and western blot analysis, the structures were found to beidentical. In addition, both preparations show comparable results in thevarious functionality tests (summarized in Table 5). Two clear majordifferences found between rh-fbg and h-Fbg were the amount of FpAphosphorylation, and the glycosylation pattern.

TABLE 5 Recombinant Fbg vs Plasma Fbg Polymerization ComparableClottability 92 vs 98% Thrombin binding 48 vs 62% Platelet aggregationComparable FXIII cross-linking Comparable FpA/B release Identical FpAphosphorylation 70 vs 30%

Example 6 Recombinant Fibrinogen has Reduced Half-Life Relative toPlasma Fibrinogen

The aim of this study was to determine the pharmacokinetics in maleWistar rats of recombinant human Fibrinogen (rhFib) (isolated from cowmilk) and plasma derived Fibrinogen (phFib).

Test System

-   -   Test system: Rat: Wistar Wu    -   Source: Charles River Laboratories, Sulzfeld, Germany    -   Total number of animals: 24 males    -   Number/group: 3-5    -   Age at start treatment: 10-15 weeks        Test Substance rhFib    -   Identification: recombinant human Fibrinogen    -   Description: isolated from cow milk by precipitation    -   Purity: ˜85%    -   Composition: 0.5 ml/vial frozen solution in 20 mM NaCitrate        pH7.0 and 0.15M NaCl    -   Concentration: 15.6 mg/ml (A280)    -   Storage: −80° C.    -   Stability: Thawed and undiluted: 24 h, 4° C.        Test Substance phFib.    -   Identification: plasma derived Fibrinogen    -   Description: isolated from human plasma by precipitation    -   Batch number: LNB-IV-01-01/36 p36    -   Purity: ˜90%    -   Composition: 0.5 ml/vial frozen solution in 20 mM NaCitrate        pH7.0 and 0.15M NaCl    -   Concentration: 9.7 mg/ml (A280)    -   Storage: −80° C.    -   Stability: Thawed and undiluted: 24 h, 4° C.        Test Method    -   Method: Intravenous injection into the tail vein or vena cava or        vena penis.    -   Dose level: See below (under: Allocation)    -   Dose volume: ˜4 ml/kg    -   Dosing speed: ˜1 ml/10 sec        Allocation of Test Groups

TABLE 6 Dose level Number Test Compound mg/ of Group substance or Batchkg animals Animal numbers 1 PBS N.A. N.A. 3 53.2 54.1 54.2 2 rhFib Prec.Cow 4 3 52.1 52.2 53.1 milk #204 3 phFib Prec. human 4 3 55.1 55.2 56.1plasmaTreatment of Test and Control Animals

-   -   Rats were anaesthetized by subcutaneous injection of 2.7 ml/kg        of hypnorm/midazolam (end concentration: fentanylcitrate: 0.08        mg/ml; fluanison: 2.5 mg/ml; midazolam 1.25 mg/ml) and the        abdomen was opened.    -   The test items were injected via the tail vein or vena cava or        vena penis. At the indicated times, blood samples of        approximately 0.2 ml were taken from the inferior vena cava and        transferred to eppendorff vials with 10 ml of 0.5 M EDTA in PBS.    -   The samples were centrifuged for 5 min at 3500×g and 100 ml        plasma of each sample is stored at −20° C. upon analysis.        Blood Sampling    -   Blood samples were taken on each of the following time points:    -   −5 (pre-dose), 2, 5, 10, 15, 20, 30, 45 and 60 minutes after end        of Fib dosing        Assay    -   The concentration of human Fib in the plasma samples was        determined using an ELISA for the detection of rhFib.

PHARMACOKINETIC EVALUATION 1. ERC Elimination Rate Constant 2. T½Half-life 3. AUC 0-t Area under the curve, from 0 till the lastquantifiable point 4. AUC 0-inf Area under the curve, from 0 tillinfinityPlasma Clearance Results

TABLE 7 Overview of tested substances, body weight Test inj dose Ratweight Substance Batch mg/kg no g Group 1 PBS N.A. N.A. 53.2 213 N.A.54.1 210 N.A. 54.2 218 Group 2 rhFib Prec. from 4 52.1 218 cowmilk #2044 52.2 209 4 53.1 211 Group 3 phFib Prec. From 4 55.1 265 human plasma 455.2 299 4 56.1 275

TABLE 8 Overview of recording of data of analysis Test inj doseSubstance Batch U/kg Rat no Group 1 PBS N.A. N.A. 53.2 N.A. 54.1 N.A.54.2 Group 2 rhFib Prec. from cowmilk #204 4 52.1 4 52.2 4 53.1 Group 3phFib Pcec. From human plasma 4 55.1 4 55.2 4 56.1Clearance Data

TABLE 9 Clearance data of rh-Fibrinogen Rat 1 Rat 2 Rat 3 Rat No 52.152.2 53.1 Weight 218 209 211 Time (min) ug/ml ug/ml ug/ml Mean st dev  0 2 47 30 32 36 9  5 42 32 34 36 5 10 34 33 28 32 3 15 30 30 25 28 3 2026 19 17 21 5 30 24  7 12 14 9 45 19 12 11 14 4 60 17 15  9 14 4

TABLE 10 Clearance data of ph-Fibrinogen Rat 1 Rat 2 Rat 3 Rat No 55.155.2 56.1 Weight 265 299 275 Time (min) ug/ml ug/ml ug/ml Mean st dev  0 2 143 128 104 125 20  5 131 125 104 120 14 10 163 128 106 132 29 15 16255 118 112 54 20 141 41 94  92 50 30 132 83 90 102 27 45 133 83 97 10426 60 138 83 87 103 31Pharmacokinetic Results

TABLE 11 Pharmacokinetic data of rh-Fibrinogen Rat 1 Rat 2 Rat 3 Rat No52.1 52.2 53.1 Mean st dev ERC 0.017 0.019 0.024 0.020 0.004 T½ 41 36 2835 6 AUC0-t 1466 1011 959 1145 279 AUC0-inf 2472 1793 1327 1864 576

TABLE 12 Pharmacokinetic data of ph-Fibrinogen Rat 1 Rat 2 Rat 3 Rat No55.1 55.2 56.1 Mean st dev ERC 0.002 0.006 0.003 0.004 0.002 T½ 444 120202 255 168 AUC0-t 8101 4820 5630 6183 1709 AUC0-inf 96507 19228 3099748911 41638

TABLE 13 Pharmacokinetical parameter overview Group 1 Group 2 Group 3Control rh-Fibrinogen Plasma Fibrinogen Parameter Mean  STD Mean  STDMean  STD ERC N.D.  0.020 ± 0.004 0.004 ± 0.002 T½ N.D. 35 ± 6 255 ± 168AUC_(0-t) N.D. 1145 ± 279 6183 ± 1709 AUC_(0-∞) N.D. 1864 ± 576 48911 ±41638

TABLE 14 T-test on half-life and AUC0-inf data Half-life AUC0-inf rH-FbgpH-Fbg rH-Fbg pH-Fbg Mean 35 255 1864 48911 Variance 41 28336 3318541733703590 Observations 3 3 3 3 Pooled Variance 14189 9E+08 Hypothesized0 0 Mean Difference Df 4 4 t Stat −2.27 −1.96 P(T <= t) one-tail 0.040.06 t Critical one-tail 2.13 2.13 P(T <= t) two-tail 0.09 0.12 tCritical two-tail 2.78 2.78

The data are summarized in FIG. 14. There is a significant difference inthe half-life of rh-Fibrinogen compared to plasma fibrinogen. Allpublications, patents and patent applications cited are hereinincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent and patent applicationwas specifically and individually indicated to be incorporated byreference in its entirety for all purposes. Unless otherwise apparentfrom the context, any step, element, embodiment, feature or aspect ofthe present application can be combined with any other step, element,embodiment, feature or aspect.

The invention claimed is:
 1. A method of treating or effectingprophylaxis in a patient who is bleeding or is at risk of bleeding,comprising administering a dose of recombinant fibrinogen having areduced half-life relative to natural human plasma fibrinogen to thepatient, wherein the peak plasma concentration of recombinant fibrinogenis greater than 1 g/L.
 2. The method according to claim 1, wherein therecombinant fibrinogen is under-sialyated.
 3. The method according toclaim 1, wherein the recombinant fibrinogen is recombinant humanfibrinogen.
 4. The method according to claim 1, wherein the half-life ofthe recombinant fibrinogen is reduced by a factor of at least 50%relative to that of human plasma fibrinogen.
 5. The method according toclaim 1, wherein the dose is greater than 1 g.
 6. The method accordingto claim 1, wherein the dose delivers a peak plasma concentration ofrecombinant and endogenous fibrinogen greater than 2 g/L.
 7. The methodaccording to claim 1, wherein the bleeding results from an acutedisorder, a traumatic injury, surgery, or an inherited disorder.
 8. Amethod of treating or effecting prophylaxis in a patient who is bleedingor at risk of bleeding, comprising administering fibrinogen having areduced half-life relative to natural human plasma fibrinogen to thepatient, wherein the treatment is in a regime sufficient to achieve apeak concentration of fibrinogen in plasma including administered andendogenous fibrinogen that is within or above normal levels, and whereinthe plasma concentration declines to normal or below normal levelswithin three days of first administration.
 9. The method according toclaim 8, wherein the peak plasma concentration of fibrinogen includingadministered and endogenous fibrinogen is above 2 g/L.
 10. The methodaccording to claim 8, wherein the peak plasma concentration offibrinogen including administered and endogenous fibrinogen is within2-4 g/L and the plasma concentration of fibrinogen declines below 2 g/Lwithin one day.
 11. The method according to claim 8, wherein the regimeis such that fibrinogen is administered once or at multiple timesoccurring within a period of no more than two hours.
 12. The methodaccording to claim 8, wherein the regime is such that fibrinogen isadministered multiple times at intervals no greater than two days. 13.The method of claim 4, wherein the half life of the recombinantfibrinogen is less than one day.
 14. The method of claim 7, wherein thebleeding results from an inherited deficiency in a coagulation protein.